DESCRIPTION: To better understand the mechanisms of the pathogenesis of tuberculosis, a more rapidly growing Mycobacterium marinum will be used as a model system to isolate and characterize virulence factors. Recently developed genetic systems for transformation, random transposition and homologous recombination will be exploited to isolate mutants compromised in virulence for entrance into and growth within macrophages. A recently developed technique, signature-tagged transposon mutagenesis, will be employed for the screening of avirulent transposon mutants compromised in virulence in the frog species Rana pipiens and in replication within cultured macrophage-like cells. As an independent approach to identifying genes encoding virulence factors, promoters with enhanced activity within the host cell milieu will be isolated using the green fluorescent protein (GFP) as a reporter gene. Transcriptional fusions will be made between random M. marinum chromosomal fragments and a promoter-less GFP gene. M. marinum transformed with this fusion library will be used to infect cultured host cells and the fluorescence intensity of the intracellular bacteria assayed by FACS. Also, the bacteria with enhanced fluorescence in host cells will be sorted by FACS, and a molecular analysis of the responsible promoters will be undertaken. Finally, M. marinum trafficking and subcellular localization will be examined by microscopic techniques. The M. marinum expressing the GFP will be studied with confocal microscopy. Immunoelectron microscopy will be used to complement these studies. Mutants compromised in virulence will be examined with these techniques as well.